Early recognition of pathogens and genetic diseases, and susceptibility and/or predisposition thereto is vitally important in healthcare and, at least in part, depends on the ability to detect nucleic acids with accuracy and sensitivity. Not surprisingly, DNA and RNA detection methods are now routinely used for forensic, paternity, military, environmental and other testing applications. Optimally, the tests must be able to generate a detectable signal from samples that contain but a few copies of a nucleic acid of interest. Accordingly, nucleic acid amplification and detection technologies are of particular interest and importance.
PCR and LCR. PCR. The polymerase chain reaction (PCR) is by far the most widely used approach for increasing the concentration of a segment of target sequence in a mixture of DNA without cloning or purification (see, e.g., U.S. Pat. Nos. 4,683,195 and 4,683,202 to Mullis K. B., 1987). Briefly, PCR employs two oligonucleotide primers which are (i) complementary to opposite strands of a double-stranded target sequence and (ii) designed to bind (hybridize) to the respective target sequence such that extension of one primer with a DNA polymerase generates a template strand for the other primer. The DNA strands in PCR are separated by denaturation, in the presence of primers, at elevated temperatures (e.g., denaturation stage at >90° C.), followed by primer hybridization at an annealing temperature (e.g., annealing stage at ˜55-65° C.) and primer extension. Because the thermostable DNA polymerases typically used in PCR exhibit maximum activity at temperatures of about 72-75° C., the primer extension may comprise a third “extension” stage at an optimal extension temperature to maximize the yield of PCR. In principle, when a quantitative yield of primer extension is achieved, the number of the DNA amplified strands is doubled after each cycle. The steps of denaturation, primer annealing, and polymerase extension can be repeated as often as needed to obtain relatively high concentrations of an amplified portion (amplicon) of the target sequence. Temperature cycling leads to rapid exponential growth of the target amplicon in accordance with the equationCn=C0(1+x)n wherein C0 is the starting DNA concentration and n is the PCR cycle number and x is the average cycle yield (0 to 1) of strand replication. Where the PCR yield is quantitative or essentially quantitative (x>0.95-0.99), the desired PCR-amplified amplicons of the target sequence become the dominant sequences in the mixture after ˜15-30 cycles, depending on the initial target DNA load. Although PCR has been widely accepted and implemented in molecular biology and DNA diagnostics, the method is yet limited by the requirement of the precise temperature-cycling apparatus, the need for hyper stable enzymes (e.g., polymerases), low multiplexing capabilities, and reaction contamination artifacts and concerns.
LCR. The ligase chain reaction (LCR) (e.g., Barany F., 1991; Wu D. Y. and Wallace R. B., 1989) is an alternative method for amplifying nucleic acids using temperature cycling. Briefly, LCR employs two pairs of self-complementary primers (or probes), the members of each pair hybridizing to respective, opposite (e.g., sense, antisense) target DNA strands, and wherein the members of each pair that hybridize to the same strand do so by hybridizing adjacent to each other on that respective strand, without gaps or mismatches. In this manner, two neighboring (adjacently hybridized) primers can be linked together by a ligase enzyme, providing a template sequence for the complementary LCR primers (also hybridizing adjacent to each other), such that repeated the cycles of denaturation, primer hybridization and ligation lead to amplification of a short segment of DNA. However, while the method has no polymerase extension requirement, there is still a requirement for cycling the between denaturation and annealing/ligation temperatures, and there is at some ability of the ligase to link two blunt-ended duplexes, leading to spontaneous, template-independent amplification that limits the applicability of LCR in detecting target nucleic acids at low concentrations.
Isothermal Amplification Technologies. Numerous attempts have been made to develop DNA amplification approaches, where the reaction does not require temperature cycling (e.g., Nucleic Acid Sequence Based Amplification (NASBA) (Davey C. and Malek L. T., 2000; Oehlenschlager F. et al, 1996), and Helicase-Dependent Amplification (HAD) (Vincent M. et al, 2004; An L. et al., 2005)). In typical isothermal amplification schemes, complementary DNA strands are separated by strand displacement during the primer extension stage, and thus require use of DNA polymerases that lack 5′-nuclease activity. Examples of such methods include Loop-Mediated Amplification (Notomi T. and Hase T., 2002; Notomi T. et al, 2000), Rolling-Circle Amplification (Lizardi P., 1998; Lizardi P. M. and Caplan M., 1998; Lizardi P. M., 2001a; Lizardi P. M., 2001b), along with various amplification methods based on use of RNA or composite RNA/DNA primers (Cleuziat P. and Mandrand B., 1998) including 5′-RNA-tailed composite primers (Kurn N., 2001; Kurn N., 2004; Kurn N., 2005) and Isothermal and Chimeric primer-initiated Amplification of Nucleic acids (ICAN) (Sagawa H. et al, 2003). All such amplification schemes are premised on having continuous DNA synthesis at a particular DNA site, which can be achieved by a number of ways including, for example, by a partial or complete by RNase H-mediated decomposition of the RNA segment of composite RNA/DNA primers; that is, after primer hybridization and extension by DNA polymerase, hydrolysis of the RNA segment of the primer promotes binding by another primer for subsequent extension and strand displacement. The RNA primer segment can be placed anywhere within the composite primer. In yet another approach, the 5′-segment of DNA primers are degraded using duplex-specific 5′-exonuclease activity (Mulrooney C. and Oultram J. D., 1999). The amplification methods that are based on partial or complete primer decomposition require a stage providing for a “fresh” primer re-annealing, wherein the remaining fragments of a previous primer have to dissociated or displaced by the fresh primer to restore the DNA priming site and support the cycling of the amplification reaction. This complicates the amplification mechanism and may slow down the reaction.
Alternatively, the primer need not be degraded, where the primer extension point can be rejuvenated via strand-specific DNA cleavage or nicking at a designated site. For example, Strand Displacement Amplification (SDA) (Walker G. T. et al, 1993; Walker G. T. et al, 1996; Fraiser M. S. et al, 1997; Walker G. T., 1998) is based on the use of a restriction enzyme to nick a hemi-modified recognition site. The method consists of a target generation process that makes copies of a target sequence that is flanked by nickable restriction sites. Amplification of these modified target sequences occurs through repeated nicking, strand displacement and extension at the restriction sites. The hemi-modified recognition sites are formed during the amplification where at least one of the four triphosphates is modified. Incorporation of modified nucleotides into amplification products blocks cleavage of the newly synthesized strands by restriction endonucleases that normally cleave both strands of double-stranded DNA.
Strand-specific cleavage of duplex DNAs is a key requirement for other reported amplification schemes (e.g. Oultram J. D. and Coutts J., 1999) and can be alternatively achieved by using recently discovered “nicking endonucleases” that cleave only one strand of a double-stranded DNA sequence. For example, Van Ness J. et al (Van Ness J. et al, 2003a; Van Ness J. et al, 2003b) suggested using the N BstNB enzyme that recognizes the 5′-GAGTC-3′ sequence, and specifically cleaves the phosphodiester link four bases downstream on this strand. In contrast to SDA, the nicking endonuclease used is naturally strand specific so that there is no need to use modified nucleotide triphosphates to preclude cleavage of the other strand. However, the 3 to 7 nucleotides-long restriction recognition motifs of such nicking endonucleases limit applicability of the approach, and adapting the approach for amplification of any desired target DNA sequence requires complicating the system design by introducing additional oligonucleotides, primers and/or pre-amplification stages (see, e.g., Van Ness J. et al, 2003a; Van Ness J. et al, 2003b; Oultram J. D. and Coutts J., 1999).
Nick Displacement Amplification (NDA) (Saba J., 2004) is a form of isothermal amplification based on strand-specific nicking and strand displacement, and is regarded as a process for synthesizing a polynucleotide with complementarity to a duplexed target polynucleotide, containing a modification which appreciably influences nicking, comprising: (a) contacting a duplexed target with a nicking agent such that the non-target strand is selectively nicked at a prescribed location; (b) extending the 3′-ended fragment adjacent the nick with a polymerase such that the nicking site is rejuvenated and the 5′-ended strand adjacent the nick is displaced; and (c) repeating steps (a) and (b) such that there are multiple cycles of nicking, extension and displacement. NDA can be performed linearly and in a fashion similar to PCR wherein two primers, complementary to opposite DNA strands, are used and wherein extension of one primer generates a template for the other primer of the pair. FIG. 4, herein, shows a schematic representation of NDA. Similar to PCR, NDA is based on the use of two ND primers, one forward and one reverse primer, which are complementary to opposite strands of double stranded target and wherein extension of one primer generates template for the other primer. Shown is a scenario where amplification is initiated by a single-stranded target DNA (sense strand). An oligonucleotide primer (forward) incorporating a nick directing modification (ND) hybridizes to the target nucleic acid (stage A). A DNA polymerase recognizes the complex, and synthesizes a complementary strand (stage B). The product of primer extension is then recognized by a nick-directed nuclease (ND nuclease) that selectively cleaves the newly synthesized DNA strand (stage C) that contains the ND modification thereby restoring the primer structure. DNA polymerase once again extends the primer while displacing the DNA strand synthesized during the previous cycle (stage D). Sequential repetition of the stages B, C and D leads to perpetual accumulation of the strand displacement products. These amplification products have an indefinite 3′-ends, but identical 5′-sequences defined by design (location) of the forward ND primer and the cleavage specificity of ND nuclease employed. The cycling polymerase extension and ND nuclease nicking at the forward ND primer generates numerous extension products with indefinite ends, which are, in turn, targets for a second ND primer (reverse primer). Perpetual amplification from the reverse primer generates multiple DNA fragments with definite ends (corresponding to the forward primer-mediated nick site). The sequence of these extension products is identical to a target DNA sequence between the primer binding sites (excluding the primer sequences).
Practicing NDA is, however, limited by the requirement for a reliable way to nick only one of two DNA strands to restore the primer function in a cycling mode. The duplexed targets may originate from the priming of a target with a modified primer; for example, nicking modification may occur within the primer sequence that hybridizes to the target, wherein such nicking modification directs nicking within or adjacent to the primer (Id). The primer modifications can be nucleotide variants or mismatched nucleotides, recognized by mutant restriction enzymes or repair endonucleases. For example, the use of deoxyinosine (dI) modification in oligonucleotide primers is proposed for use as a nick-directing agent (Id), and it has been well established in the art that the certain endonucleases that initiate repair of dI lesions (see FIG. 1 herein), such as Endonuclease V from E. coli (Endo V), selectively cleave the dI-containing strand in DNA duplex at a site located 3′ (downstream) from the lesion (see FIG. 2 herein). Endo V from E. coli was first described by Gates and Linn (Gates F. T. III and Linn S., 1977), and later extensively characterized by Yao and co-workers (Yao M. et al, 1994; Yao M. and Kow Y. W., 1994; Yao M., Kow Y. W., 1995; Yao M., Kow Y. W., 1996; Yao M., Kow Y. W., 1997). Homologs of E. coli Endo V have been identified in a wide variety of organisms including archaebacteria, eubacteria and eukaryotes. Endo V analogs were also isolated from hyperthermophiles Archaeoglobus fulgidus (Liu J. et al, 2000), Thermotoga maritima (Huang J. et al, 2001; Huang J. et al, 2002), and mice (Moe A. et al, 2003). Deamination of adenosine in natural DNAs results in a dI-dT mismatch which may be repaired according to the pathway shown in FIG. 2 herein. However, such natural dI-dT mismatch repair does not provide a substrate primer for DNA polymerase because of the adjacent dI-dT mismatch; that is, the 3′-to-5′-Exo (endo) nuclease activity (aka, proof-reading activity) of a DNA polymerase degrades the 3′-nicked strand, and once the mutated dI-base is removed DNA integrity is restored by DNA extension and ligation). However, Endo V has also been shown to cleave complexes wherein dI forms a Watson-Crick base pair with dC. This property of the repair endonuclease makes dI containing primers useful in practicing NDA (see FIG. 3 herein). Unlike the case with nicking endonucleases, the use of deoxyinosine as a nick directing modification provides ample flexibility for NDA primer design with respect to any desired target nucleic acid sequence.
Millar D. S. et al have also disclosed an isothermal amplification reaction (Millar D. S. et al, 2006), which is similar to NDA in many aspects. Briefly, two oligonucleotide primers are designed in a fashion similar to PCR to provide isothermal amplification of nucleic acids in the presence of a strand displacing DNA polymerase, wherein the primers incorporate non-regular bases. Particular aspects comprise use of an enzyme that recognizes a non-regular base in double stranded DNA, and that causes a nick or excises a base in one DNA strand, at or near the site of the non-regular base, for DNA amplification substantially without thermal cycling. Similar to the case of NDA, Millar D. S. et al disclose numerous examples of non-regular bases (e.g., inosine) and respective enzymes (e.g., Endonuclease V, and variants thereof) that recognize the non-regular bases and cleave only one strand of duplex DNA to support the amplification reaction (Id).
In NDA (Saba J., 2004; Millar D. S. et al, 2006) and other NDA-related amplification schemes (Van Ness J. et al, 2003a; Van Ness J. et al, 2003b; Oultram J. D. and Coutts J., 1999), the nick directing primers do not need to dissociate and they can stay hybridized to the target nucleic acid indefinitely during the amplification while initiating numerous cycles of extension and strand displacement. This provides advantages over amplification schemes that are based on complete or partial degradation of the primer. Developers of ICAN made an attempt to adopt this advanced NDA format wherein RNA/DNA composite primers are not degraded, but rather restored by RNase H activity (e.g. Mukai H. et al, 2003). However, an advantage in this instance comes at the cost of having amplification primers terminated by ribonucleotides that are less efficiently extended by DNA polymerases than the DNA analogs.
Despite the isothermal aspects, the isothermal amplification methods including NDA (Saba J., 2004; Millar D. S. et al, 2006) and NDA-related methods (Van Ness J. et al, 2003a; Van Ness J. et al, 2003b; Oultram J. D. and Coutts J., 1999; Mukai H. et al, 2003) are limited because of reasons discussed herein, but primarily because of the relatively low amplification efficiency or amplification rate (e.g., Millar D. S. et al, 2006),
There is therefore, a pronounced need in the art for more efficient and more rapid nucleic acid detection methods, including more efficient isothermal amplification methods that are not limited by the sequence of target nucleic acids of interest, multiplexing capabilities, choice of detection technology or requirements for post-amplification detection, sensitivity (i.e. minimum target load) and selectivity of amplification, and other factors and parameters that define the scope of the methods applicability in science and technology.